1. Field of the Invention
The invention relates to the inhibition of the Cyclin-Dependent Kinase Inhibitor (CDKI) pathway. More particularly, the invention relates to compounds and methods for inhibiting the CDKI pathway for studies of and intervention in senescence-related diseases, including degenerative diseases of the central nervous system, including Alzheimer's Disease and other dementias, as well as for studies of and intervention in cancer and viral diseases.
2. Summary of the Related Art
Cell senescence, originally defined as a series of cellular changes associated with aging, is now viewed more broadly as a signal transduction program leading to irreversible cell cycle arrest, accompanied by a distinct set of changes in the cellular phenotype (See e.g. Campisi, Cell 120: 513-522 (2005); Shay and Roninson, Oncogene 23: 2919-2933 (2004)). Senescence can be triggered by many different mechanisms including the shortening of telomeres (replicative senescence) or by other endogenous and exogenous acute and chronic stress signals, including major environmental factors, such as UV and cigarette smoke. The latter forms of telomere-independent senescence are variably referred to as accelerated senescence, STASIS (Stress or Aberrant Signaling Induced Senescence), or SIPS (Stress-Induced Premature Senescence). Regardless of the mode of induction, senescent cells develop the same general phenotype, characterized not only by permanent growth arrest but also by enlarged and flattened morphology, increased granularity, high lysosomal mass, and expression of senescence-associated endogenous β-galactosidase activity (SA-β-gal).
Dimri et al., Proc. Natl. Acad. Sci. USA 92: 9363-9367 (1995) teaches that in the human body, the phenotype of cell senescence has been detected in correlation with aging. Castro et al., Prostate 55: 30-38 (2003); Michaloglou et al., Nature 436: 720-724 (2005); and Collado et al., Nature 436: 642 (2005) teach that the phenotype of cell senescence has also been detected in pathological situations, including various pre-malignant conditions. te Poele et al., Cancer Res. 62: 1876-1883 (2002); and Roberson et al., Cancer Res. 65: 2795-2803 (2005) teach its detection in many tumors treated with chemotherapy.
In most systems of senescence that have been characterized at the molecular level, cell cycle arrest is triggered by the activation of p53, which in its turn induces a broad-specificity cyclin-dependent kinase inhibitor (CDKI) p21Waf1/Cip1/Sdi1. p21 induction causes cell cycle arrest at the onset of senescence, but p53 and p21 levels decrease at a later stage. Shay and Roninson, Oncogene 23: 2919-2933 (2004) teach that this decrease is accompanied, however, by a stable increase in another CDKI protein, p16Ink4A, which is believed to be primarily responsible for the maintenance of cell cycle arrest in senescent normal cells.
CDKI proteins act as negative regulators of the cell cycle and are therefore generally known as tumor suppressors. The induction of CDKI proteins, in particular p21, also occurs in tumor cells in the context of cancer therapy, in response to cellular damage by different classes of cancer chemotherapeutic drugs and ionizing radiation. Cell cycle arrest by CDKIs mediates the cytostatic and senescence-inducing activity of anticancer agents, one of the major components of their therapeutic effect (Roninson, Cancer Res., 11, 2705-2715). Agents that would enhance the ability of CDKI proteins to induce cell cycle arrest will therefore be useful for the chemoprevention of cancer and for increasing the therapeutic efficacy of conventional anticancer agents.
Although senescent cells do not divide, they remain fully viable, metabolically and synthetically active. It has now been recognized that senescent cells secrete a variety of factors that have a major effect on their environment. Campisi, supra teaches that secretory activities of senescent cells have been linked to carcinogenesis, skin aging, and a variety of age-related diseases. A series of studies have implicated p21 and other CDKI proteins in disease-promoting activities of senescent cells. This insight came principally from the analysis by Chang et al., Proc. Natl. Acad. Sci. USA 97: 4291-4296 (2000) of the transcriptional effects of p21, expressed in a fibroblastoid cell line from an inducible promoter. This analysis showed that p21 produces significant changes in the expression of multiple genes. Many genes are strongly and rapidly inhibited by p21, and most of these are involved in cell proliferation. Zhu et al., Cell Cycle 1: 50-58 (2002) teaches that inhibition of cell cycle progression genes by p21 is mediated by negative cis-regulatory elements in the promoters of these genes, such as CDE/CHR. The same genes are downregulated in tumor cells that undergo senescence after chemotherapeutic treatment, but Chang et al., Proc. Natl. Acad. Sci. USA 99: 389-394 (2002) teaches that p21 knockout prevents the inhibition of these genes in drug-treated cells. Hence, p21 is responsible for the inhibition of multiple cell cycle progression genes in response to DNA damage.
Chang et al., 2000, supra teaches that another general effect of p21 induction is upregulation of genes, many of which encode transmembrane proteins, secreted proteins and extracellular matrix (ECM) components. This effect of p21 is relatively slow, occurring subsequently to growth arrest and concurrently with the development of the morphological features of senescence. These genes are induced by DNA damage but p21 knockout decreases their induction (Chang et al., 2002, supra). This decrease is only partial, which can be explained by recent findings by that the majority of p21-inducible genes are also induced in response to other CDKI, p16 and p27 (see WO 03/073062). Gregory et al., Cell Cycle 1: 343-350 (2002); and Poole et al., Cell Cycle 3: 931-940 (2004) teach that gene upregulation by CDKI has been reproduced using promoter constructs of many different CDKI-inducible genes, indicating that it occurs at the level of transcription. (Perkins et al., Science 275: 523-527 (1997); Gregory et al., supra; and Poole et al., supra teach that induction of transcription by p21 is mediated in part by transcription factor NFκB and transcription cofactors of p300/CBP family, but other intermediates in the signal transduction pathway that leads to the activation of transcription in response to CDKI—the CDKI pathway—remain presently unknown (FIG. 1).
Medical significance of the induction of transcription by CDKI has been indicated by the known functions of CDKI-inducible genes (Chang et al., 2000, supra). Many CDKI-upregulated genes are associated with cell senescence and organism aging, including a group of genes implicated in age-related diseases and lifespan restriction. One of these genes is p66Shc, a mediator of oxidative stress, the knockout of which expands the lifespan of mice by about 30% (Migliaccio et al., supra). Many CDKI-induced genes play a role in age-related diseases, most notably Alzheimer's disease and amyloidosis. Thus, CDKI induce many human amyloid proteins, including Alzheimer's amyloid β precursor protein (βAPP) and serum amyloid A, implicated in amyloidosis, atherosclerosis and arthritis. CDKI also upregulate tissue transglutaminase that cross-links amyloid peptides leading to plaque formation in both Alzheimer's disease and amyloidosis. Some of CDKI-inducible genes are connective tissue growth factor and galectin-3 involved in atherosclerosis, as well as cathepsin B, fibronectin and plasminogen activator inhibitor 1, associated with arthritis. Murphy et al., J. Biol. Chem. 274: 5830-5834 (1999) teaches that several CDKI-inducible proteins are also implicated in an in vitro model of nephropathy. Remarkably, p21-null mice were found to be resistant to experimental induction of atherosclerosis (Merched and Chan, Circulation 110: 3830-3841 (2004)) and chronic renal disease (Al Douahji et al., Kidney Int. 56: 1691-1699 (1999); Megyesi et al., Proc. Natl. Acad. Sci. USA 96: 10830-10835 (1999).
In addition to their effect on cellular genes, CDKI stimulate the promoters of many human viruses, such as HIV-1, cytomegalovirus, adenovirus and SV40. Since many viruses induce p21 expression in infected cells, this effect suggests that promoter stimulation by CDKI may promote viral infections (Poole et al., supra).
Strong associations for CDKI-inducible genes have also been found in cancer. In particular, p21 expression activates the genes for many growth factors, inhibitors of apoptosis, angiogenic factors, and invasion-promoting proteases. In accordance with these changes in gene expression, Chang et al., 2000, supra teaches that p21-arrested cells show paracrine mitogenic and anti-apoptotic activities in coculture assays. Krtolica et al., Proc. Natl. Acad. Sci. USA 98: 12072-12077 (2001) teaches that paracrine tumor-promoting activities were demonstrated both in vitro and in vivo in CDKI-expressing normal senescent fibroblasts, which express p21 and p16. Importantly, senescent fibroblasts possess the characteristic pro-carcinogenic activity that has long been identified with tumor-associated stromal fibroblasts. Furthermore, all the experimental treatments shown to endow fibroblasts with tumor-promoting paracrine activities also induce CDKI, suggesting that the CDKI pathway could be the key mediator of pro-carcinogenic activity of stromal fibroblasts (Roninson, Cancer Lett. 179: 1-14 (2002)).
CDKI expression mediates cell cycle arrest not only in the program of senescence but also in numerous other situations, such as transient checkpoint arrest in response to different forms of damage, contact inhibition, and terminal differentiation. Hence, the CDKI pathway, which leads to the activation of multiple disease-promoting genes, is activated not only in cell senescence but also in many other physiological situations. As a result, CDKI-responsive gene products are expected to accumulate over the lifetime, contributing to the development of Alzheimer's disease, amyloidosis, atherosclerosis, arthritis, renal disease, viral diseases, including HIV/AIDS and cancer.
The effects of CDKIs are usually considered in light of their inhibition of cyclin-dependent kinases (CDKs), a family of serine/threonine kinases comprising 21 members in the human genome, which act in a complex with regulatory cyclin proteins. The best-known CDKs (CDK1, CDK2, CDK4, CDK6) are required for transitions between different phases of the cell cycle, but many other CDKs function as regulators of transcription or RNA processing rather than the cell cycle (Malumbres et al., 2009). Among the latter, of special relevance to the instant invention are CDK8 and a closely related CDK19 (80% overall identity, 98% identity in the ATP pocket). These CDKs, coupled with Cyclin C, are alternative components of a regulatory module of the Mediator complex that connects transcriptional regulators with RNA polymerase II to initiate transcription (Sato et al., 2004). CDK19 has been also called CDC2L6 and, confusingly, CDK11, but the name CDK11 is more often applied to two other proteins, presently known as CDK11A and CDK11B (Malumbres et al., 2009). CDK8 has been the subject of great attention in recent years and was identified as playing an important role in cancer (reviewed in Firestein and Hahn, 2009). CDK8 knockdown and knockout studies showed that it is not needed for cell growth but is required for early embryonic development (Westerling et al., 2007). CDK8 has been associated with processes involved in senescence and damage response: it regulates Smad transcriptional activation and turnover in BMP and TGF-β pathways (Alarcon et al., 2009) and acts as a stimulus-specific positive coregulator of p53 target genes (Donner et al., 2007). CDK8 has been identified as an oncogene amplified in ˜50% of colon cancers, acting as a positive regulator of β-catenin, a transcription factor that plays a central role in colon carcinogenesis (Firestein et al., 2008; Morris et al., 2008). CDK8 has not yet been implicated in human diseases other than cancer.
In contrast to CDK8, little is known about CDK19, which substitutes for CDK8 in the corresponding Mediator modules but may have an opposite effect on the regulation of transcription. In particular, the study of Tsutsui et al. (2008), which refers to CDK19 as CDK11, reports that CDK19 acts as a negative regulator of viral activator VP16-dependent transcription, in contrast to CDK8 that acts as a positive regulator in this system. Pohlner and Von der Kammer (US patent publication 2009/00047274 A1) report that CDK19 (called there CDC2L6) is upregulated at the level of mRNA expression in the inferior temporal cortex and in the frontal cortex of the brains of patients with Alzheimer's disease (AD) relative to the brains of control individuals. It speculates that CDK19 (CDC2L6) could be used as a drug target for the treatment of AD but offers no evidence for that contention, aside from its overexpression in this disease.
US Patent Application Publication No. 20080033000 discloses a series of structurally related compounds, which inhibit the induction of all the tested genes by CDKI and also reverse CDKI-induced transcription. Those molecules showed little or no cytotoxicity in normal cells. As such, those molecules provided a promising starting point for developing useful new compounds and methods for inhibiting the CDKI pathway. Greater potency of such molecules is still needed.
A compound inhibiting CDK8 and CDK19 preferentially to other CDKs has been previously reported. This inhibitor is a steroidal alkaloid cortistatin A, isolated from the marine sponge Corticium simplex. The only biological properties reported for cortistatin A are its strong and highly selective antiproliferative activity against human umbilical vein endothelial cells (HUVECs) and its ability to inhibit vascular endothelial growth factor (VEGF)-induced migration and tubular formation of HUVECs (Aoki et al., 2007). In particular, cortistatin A inhibited the proliferation of HUVECs with IC50 of 1.8 nm, whereas its antiproliferative activity against several other types of human cells was 6-7 μM, or >3,000-fold higher than for HUVECs. Cee et al. (2009) tested cortistatin A at 10 mm for the ability to inhibit a panel of 402 kinases (KinomeScan, Ambit Biosciences, San Diego, Calif.), and found that the strongest inhibition was observed for ROCK II (Percent of Control (POC)=0), CDK19 (termed there CDK11) (POC=0.1), and CDK8 (POC=0.95). The binding constants (Kd) were determined for CDK19 (Kd=10 nm), CDK8 (Kd=17 nm), and ROCK I and II (Kd=250 nm and 220 nm, respectively). It has been unknown whether the selective antiproliferative effect of cortistatin A against HUVECs is due to the inhibition of CDK19, CDK8 or ROCK (Cee et al., 2009). Based on its selective effect on endothelial cells, cortistatin A was proposed as a new type of anti-angiogenesis agent for the treatment of cancer (Aoki et al., 2007). However, the usefulness of anti-angiogenic agents for long-term therapy of chronic diseases other than cancer is doubtful, since clinical experience with angiogenesis inhibitors such as bevacizumab (Avastin), has revealed severe side effects (Grothey and Galanis, 2009), such as gastrointestinal perforation, inhibition of wound healing, and fatal pulmonary hemorrhage. Therefore, if any inhibitors of CDK8 could be found that don't have the strong anti-proliferative effect on endothelial cells, characteristic for cortistatin A, such inhibitors could be used for many clinical applications where angiogenesis inhibitors are precluded by their side effects.
There is, therefore, a need for more potent compounds and methods for inhibiting the CDKI pathway which may have a variety of clinical applications in chemoprevention and therapy of different age-related diseases. There is also a need for more potent compounds and methods for inhibiting CDKI pathway-mediated paracrine support for cancer development by senescent fibroblasts and for inhibiting viral replication. In addition there is a need for potent inhibitors of CDK8 that do not have strong anti-proliferative effects on endothelial cells.